Our lab is focused on improving the understanding of the regulation of hematopoietic stem cells. Recently we identified a critical role for the de novo DNA methyltransferase, DNMT3A, in the maintenance of normal hematopoiesis. Deletion of Dnmt3a in a murine model led to dramatically increased stem cell self-renewal and blocked differentiation. Our findings are relevant to human disease, as DNMT3A is frequently the founding genetic lesion in a variety of hematologic malignancies and is the most commonly mutated gene in age-related clonal hematopoiesis. Our studies and these findings in human diseases indicate that stem cells in which DNMT3A function is lost have a competitive advantage, allowing for aberrant clonal expansion. The parent award associated with this supplement aims to gain insight into the mechanisms by which DNMT3A mutations confer an advantage to HSCs and contribute to clonal expansion as well as to the development of MDS and leukemia in the general population. In this proposal, we aim to similarly study the mechanisms by which hematopoietic stem cells in some children with Down syndrome (DS) gain a competitive advantage, undergo clonal expansion and, in some, ultimate malignant transformation. The clonally expanded populations frequently harbor mutations of regulators of higher order chromatin structure, including mutations of DNMT3A, cohesin complex members and mutations/deletions of CTCF. This indicates many of the same mechanisms driving clonal expansion in age related clonal hematopoiesis, may be at work in children with DS. The work we propose here will build upon our ongoing work in non-DS clonal hematopoiesis and provide new insight into the underlying aberrations of clonal expansion in DS. In addition to being well within the scope of the parent NIDDK award, the work we propose here directly addresses INCLUDE Project Component 1 as it is a basic science investigation highly relevant to DS, with a high likelihood of positively impacting the health of a large percentage of children with DS. The studies we will conduct includes development of novel murine models of the clonal DS hematopoietic disorder, TAM, and epigenetic and transcriptomic profiling in these model DS hematopoietic systems, both topics of emphasis under the INCLUDE Project. Our findings could identify key targets for therapeutic intervention, paving the way for future clinical trials for children with DS. This work is also well aligned with the overarching goal of the Hematologic Disease research program of the NIDDK, which aims to improve the understanding of the basic cellular and molecular mechanisms underlying the production and function of blood cells. In summary, we aim to leverage our lab?s expertise in the study of hematopoiesis, extending our investigations of mechanisms of clonal expansion in non-DS individuals to study the clonal hematologic anomalies prevalent in children with DS.